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High throughput screening method for high-yield tylosin strain based on flow cytometry

A technology of flow cytometry and tylosin, which is applied in the field of high-throughput screening of high-yield tylosin strains, can solve the problems of dead cells, long screening cycle, heavy workload, etc. Improvement of speed and screening efficiency, effect of increasing growth rate

Inactive Publication Date: 2016-12-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current classical mutagenesis combined with high-throughput screening method may lead to the failure of subsequent culture due to the existence of a large number of dead cells in the classical mutagenesis. At the same time, the strains after the classical mutagenesis must be further isolated, purified, and expanded for subsequent enzyme labeling. There are problems such as long screening period and heavy workload in the early stage for high-throughput screening such as plate screening

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 ARTP or other physical and chemical mutagenesis to obtain different active spores:

[0039] The starting strain Streptomyces freundii slant strain was cultured at 29° C. for 6 days until the spores matured and the slant colonies were milky white. Scrape an appropriate amount of mature slant spores into a test tube filled with 2mL of PBS buffer solution, vibrate with a shaker to make a bacterial suspension, and filter with sterile filter paper. Draw 10 μL of a suitable concentration of bacterial suspension and evenly spread it on the surface of a sterile slide, place the slide on the stage, and use the ARTP mutagenesis instrument for mutagenesis. Conditions: The incident power is 100W, the helium flow rate is 10SLM, and spores with different activities are obtained by irradiating for a certain period of time. Other physical and chemical mutagenesis can also obtain spores with different activities.

Embodiment 2

[0040] Embodiment 2 PI dyestuff to Streptomyces flexneri spore staining:

[0041] After the sample mutagenesis treatment is completed, use sterile tweezers to place the slide into the EP tube filled with PBS (pH 7.4) buffer solution, shake it well for 1 min, and the bacteria attached to the slide are completely eluted into the liquid PBS . Filter with filter paper, centrifuge at 5000×g for 5 min, discard the supernatant, and add PBS to resuspend. Repeat this operation step 2 to 3 times to obtain a pure spore suspension of Streptomyces flexneri. Dilute the pure S. flexneri spore suspension to a concentration of 10 with PBS 5 ~106 spores / mL. Take an equal volume of Streptomyces freundii spore suspension and mix with 10 μg / mL PI dye, and place in a 4°C refrigerator for 20 minutes in the dark.

Embodiment 3

[0042] Example 3 Flow Cytometry Detection and Sorting:

[0043] Detection should establish a control test to remove non-specific binding or background. A sample not labeled with any fluorescein was used as a negative control, representing the background fluorescent signal from the cells. Adjust the base voltage with a negative control to set the negative and positive population boundaries. A completely inactive, PI stain-bound sample was used as a positive control to determine negative and positive spore populations. Sample testing was performed after the conditions were fixed. The stained Streptomyces flexneri spore suspension was taken, re-filtered, diluted to a certain multiple, and analyzed by a flow cytometer (MoFlo XDP). The obtained data were analyzed with Summit 5.4 software. According to PI emission red fluorescence wavelength, with FL3 channel ((610±15)nm) detection. By adjusting FSC, SSC, fluorescent channel coordinate parameters, voltage and threshold paramete...

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Abstract

The invention discloses a high throughput screening method for a high-yield tylosin strain based on a flow cytometry, belonging to the technical field of high throughput screening. According to the method, a PI dyeing method is carried out on induced-mutation spores with different activities, active spore regions are correctly distinguished by adjusting and optimizing parameters including FSC, SSC, a voltage threshold and the like, and the interference blended with a background in flow cytometry detection caused by over-small volumes of the spores is eliminated; by carrying out a solid-liquid combined high throughput culture scheme on the separated viable spores, the pore plate growth rate of streptomyces fradiae is effectively increased; and activated streptomyces fradiae is separated based on the flow cytometry, and a novel effective manner is provided for the high throughput screening of the streptomyces fradiae.

Description

technical field [0001] The invention relates to a high-throughput screening method for high-throughput tylosin-producing strains based on flow cytometry, and belongs to the technical field of high-throughput screening. Background technique [0002] Tylosin (Tylosin), also known as Tylosin and Tylomycin, is a 16-membered macrolide antibiotic. It is a high-efficiency and non-residue antibacterial growth-promoting agent for poultry and livestock. It is widely used in feed , Veterinary drug additives. The industrial tylosin-producing bacteria is Streptomyces fradiae. Tylosin is mainly composed of four components: tylosin A, mycosyl tylosin B, macrocin C and resamectin D, and component A has the strongest biological activity. The biosynthetic process of telosin is very complex, and the specific biosynthetic pathway has not yet been elucidated. Through molecular biology and genetic engineering techniques, the biosynthetic gene clusters for transforming tylosin are still being e...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12N1/20C12R1/54
CPCC12N15/01C12N1/20
Inventor 周景文陈坚王得明张庆辉王丽丽堵国成曹晓梅
Owner JIANGNAN UNIV
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